Composition and method for treating graft-versus-host disease

ABSTRACT

Compositions and methods are provided for prevention and clinical treatment of various forms of graft-versus-host disease (GVHD) by using inhibitors of adenosine deaminase (ADA). In particular, various formulations and dosing regimens of ADA inhibitors such as pentostatin are provided for the treatment of humans in vivo as well as for ex vivo conditioning of organ transplants in order to specifically suppress T-lymphocyte mediated immune responses while minimizing systemic toxicity of the drug.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to compositions and methods for thetreatment of graft-versus-host disease, and more specifically to theadministration of inhibitors of adenosine deaminase such as pentostatinand analogs and derivatives thereof.

[0003] 2. Description of Related Art

[0004] For dysfunctional and/or diseased organs of the body, besidestherapeutic invention with drugs, organ transplantation is analternative, sometimes the last resort in the treatment of the patient.Particularly for patients with leukemia, end-stage renal, cardiac,pulmonary or hepatic failure, organ transplantation is quite commonlyused in the treatment. For example, allografts (organ grafts harvestedfrom donors other than the patient him/herself or host/recipient of thegraft) of various types, e.g. kidney, heart, lung, liver, bone marrow,pancreas, cornea, small intestine and skin (e.g. epidermal sheets) arecurrently routinely performed. Xenografts (organ grafts harvested fromnon-human animals), such as porcine heart valves, are also being usedclinically to replace their dysfunctional human counterparts.

[0005] To ensure successful organ transplantation, it is desirable toobtain the graft from the patient's identical twin or his/her immediatefamily member. This is because organ transplants evoke a variety ofimmune responses in the host, which results in rejection of the graftand graft-versus-host disease (hereinafter, referred to as “GVHD”).

[0006] The immune response is primarily triggered by T cells throughrecognition of alloantigens, and the major targets in transplantrejection are non-self allelic forms of class I and class II MajorHistocompatibility Complex (MHC) antigens. In acute rejection, donor'santigen-presenting cells such as dendritic cells and monocytes migratefrom the allograft to the regional lymph nodes, where they arerecognized as foreign by the recipient's CD4⁺ T_(H) cells, stimulatingT_(H) cell proliferation. Following T_(H) cells proliferation, apopulation of effector cells (including cytotoxic CD8⁺ T cells and CD4⁺T cells) is generated, which migrates and infiltrates to the graft andmediates graft rejection (Noelle et al. (1991) FASEB 5(13):2770).

[0007] Whereas acute rejection is a T cell-dependent process, a broadarray of effector mechanisms participates in graft destruction. Throughthe release of cytokines and cell-to-cell interactions, a diverseassembly of lymphocytes including CD4⁺ T cells, CD8⁺ cytotoxic T cells,antibody-forming B cells and other proinflammatory leukocytes, isrecruited into the anti-allograft response. Antigen-presenting graftcells are destroyed directly by cytotoxic CD8⁺ T cells. Activated CD4⁺ Tcells produce interleukin-2 (hereinafter, referred to as “IL-2”), whichis essential to the activation of both CD8⁺ T cells and B cells.Additionally, CD4⁺ T cells produce other cytokines such as IFN-γ andIL-4 that also contribute to the destruction of allograft. Furthermore,interferon-γ (hereinafter, referred to as “IFN-γ”) induces increasedexpression of class I and class II MHC molecules on graft tissue, whichis more readily attacked by alloreactive effector cells. IFN-γ enhancesmacrophage activity and affects many inflammatory cells leading todelayed-type-hypersensitivity reaction and inflammation causingnonspecific damage to the graft. These reactions appear to be theprimary cause of the early acute rejection that may occur within thefirst few weeks after transplant. If untreated, acute rejectionprogresses to a rapid and severe process that causes destruction of thetransplant within a few days.

[0008] On the other hand, when a T-lymphocyte from the donor recognizesthe differences based on a set of genetic markers, generally referred toas human leukocyte antigens (HLA), and it starts to attack the new body,i.e., the patient's body. Although most patients and donors are matchedas closely as possible for HLA markers. Many minor markers, however,differ between donors and patients except when the patient and donor areidentical twins. Before a transplant, extensive typing of the donor andrecipient is performed to make sure that the donor and recipient arevery close immunologically. Despite this typing there are immunologicaldifferences that cannot be detected and that the T-lymphocytes in thedonor graft are capable of detecting. As a result, the donorT-lymphocytes start to attack the patient's body and cause GVHD.

[0009] There are two forms of GVHD: the acute and chronic GVHD. AcuteGVHD usually occurs within the first three months following atransplant. T-cells present in the donor's bone marrow at the time oftransplant attack the patient's skin, liver, stomach, and/or intestines.The earliest signs of acute GVHD are usually a skin rash that appears onthe hand, feet and face. Other than blistering skin, patients withsevere GVHD also develop large amounts of watery or bloody diarrhea withcramping due to the donor's T-cells' attack on the stomach andintestines. Jaundice (yellowing of the skin and eyes) is the usualindication that GVHD disease involves the liver. The more organsinvolved and the worse the symptoms, the worse the GVHD disease.

[0010] In the case of bone marrow transplantation, in particular, GVHDis another obstacle to survival of transplanted patients. Storb (1984)“Pathophysiology and prevention of graft-versus-host disease.” InAdvances in Immunobiology: Blood cell antigens and bone marrowtransplantation, McCullogh and Sandier, editors, Alan, Inc., N.Y.,p.337. A large proportion of GVHD-afflicted individuals dies as a resultof GVHD. Weiden et al. (1980) “Graft-versus-host disease in allogeneicmarrow transplantation”, in Biology of Bone-Marrow Transplantation, Galeand Fox, editors, Academic Press, N.Y., p37.

[0011] To protect patients from such fatal damages, variousimmunosuppressive agents have been employed. Currently, allograftrejection is controlled using immunosuppressive agents such ascyclosporin A, azathioprine, corticosteroids including prednisone, andmethylprednisolone, cyclophosphamide, and FK506. Cyclosporin A, the mostpowerful and most frequently used immunosuppressant, revolutionized thefield of organ transplant surgery. Other immunosuppressive agents suchas FK506, rapamycin, mycophenolic acid, 15-deoxyspergualin, mimoribine,misoprostol, OKT3 and anti-IL-2 receptor antibodies, have been used inthe treatment and/or prevention of organ transplantation rejection.Briggs, Immunology letters, 29(1-2), 89-94, 1991; FASEB 3:3411, 1989.Although the development of new immunosuppressive drugs has led tosubstantial improvement in the survival of patients, these drugs areassociated with a high incidence of side effects such as nephrotoxicityand/or hepatotoxicity.

[0012] For example, cyclosporin A has associated toxicities and sideeffects when used even at therapeutic doses. Although FK506 is about 10to 100 times more potent than cyclosporin A in inhibitingactivation-induced IL-2 transcription in vitro and graft rejection invivo, it also shows side effects such as neurotoxicity andnephrotoxicity. Thus, there still exists the need for treatment andprophylaxis for GVHD with improved toxicity profiles.

SUMMARY OF THE INVENTION

[0013] Compositions and methods are provided for prevention and clinicaltreatment of various forms of graft-versus-host disease (GVHD) by usinginhibitors of adenosine deaminase (ADA). In particular, novelformulations and dosing regimens of ADA inhibitors such as pentostatinare provided for the treatment of humans in vivo as well as for ex vivoconditioning of organ transplants in order to specifically suppressT-lymphocyte mediated immune responses while minimizing systemictoxicity of the drug.

[0014] In one aspect, a method is provided for treating a patient havinggraft-versus-host disease. The method comprises: administering to thepatient an adenosine deaminase (ADA) inhibitor in a pharmaceuticallyeffective amount. Examples of the adenosine deaminase inhibitor include,but are not limited to, pentostatin, fludarabine monophosphate, andcladribine. The ADA inhibitor may be administered orally or parenterally(e.g., via intravenous infusion or injection) to the patient.

[0015] The patient may have acute or chronic graft-versus-host disease,and may have also failed at least one immunosuppressive regimen such asa regimen including steroids (e.g., prednisone and methylprednisolone),cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine, anddaclizumab.

[0016] In one embodiment, the method is used to treat hematopoietic stemcell transplant (HSCT) patients manifesting grade 2 or greater acuteGVHD, who have failed to respond to treatment with at least 2 mg/Kg ofmethylprednisolone or equivalent corticosteroid or other salvagetherapy. For example, the HSCT patient may be treated with pentostatinat 0.25-1 mg/m²/day as a 20 minute intravenous (IV) infusion on days 1,2 and 3.

[0017] The method may further comprise: monitoring the improvement ofthe GVHD symptoms in the skin, mouth, fascia, and liver. Treatment withpentostatin may be repeated to further reduce the symptoms or to preventrecurrence of the disease.

[0018] In another embodiment, the method is used to treatsteroid-refractory chronic graft vs host disease (cGVHD). For example,recipients of allogeneic HSCT developing cGVHD who have failed torespond to treatment with at least 2 mg/Kg of methylprednisolone orequivalent corticosteroid or other salvage therapy may be treated withpentostatin. For example, pentostatin may be orally administrated to thechronic GVHD patient at a dose between about 1-10 mg/m², preferablybetween about 2-6 mg/m², and more preferably between about 2-4 mg/m²each day for 3 consecutive days each month.

[0019] In another aspect, a method is provided for preventing orreducing the risk of developing graft-versus-host disease in a recipientof an organ or tissue transplant. The method comprises: administering tothe transplant recipient an adenosine deaminase (ADA) inhibitor in apharmaceutically effective amount within a predetermined time windowbefore or after the transplantation. Examples of the ADA inhibitorinclude, but are not limited to, pentostatin, fludarabine monophosphate,and cladribine. The ADA inhibitor may be administered orally orparenterally (e.g., via intravenous infusion or injection) to therecipient of organ transplatation.

[0020] In one embodiment, pentostatin is administered orally to thetransplant recipient 3 or 2 days before the transplantation.Alternatively, pentostatin may be administered to the transplantrecipient by IV infusion at a dose lower than 2 mg/m², preferably at adose lower than 1 mg/m².

[0021] In a variation of the embodiment, the transplant recipient istransplanted with hematopoietic stem cells and treated in amyeloablative conditioning regimen. The recipient may be treated withpentostatin via oral administration at about 0.5-2.0 mg/m² on days −14,−13, −12 and −3, −2, −1 with high dose cyclophosphamide and/or busulfanand/or melphalan and/or 1200-1800 cGy irradiation prior to stem cellinfusion. Post transplantation the recipient may be continuously treatedwith pentostatin, on days +8 and +15 preferably iv at a lower dose suchas 1-2 mg/m².

[0022] In another embodiment, pentostatin may be administered to atransplant recipient after the transplantation. For example, forstandard (i.e., myeloablative) transplant or non-myeloablative stem celltransplant (NST) where pentostatin is not used in the conditioningregimen, pentostatin is administered to the transplant recipient at0.5-1.5 mg/m²/day on days +8, +15, +22 and +30 following stem cellinfusion.

[0023] The above methods may further comprise: administering to the GVHDpatient or the transplant recipient an immunosuppressive agent selectedfrom the group consisting of prednisone, methylprednisolone,cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine,Daclizumab, Infliximab, MEDI-205, abx-cbl and ATG.

[0024] In yet another aspect, a method is provided for ex vivo or invitro treatment of blood derived cells, bone marrow transplants, orother organ transplants. The method comprises: treating a tissue ororgan transplant with an ADA inhibitor in an effective amount such thatactivity of T-lymphocytes therein is substantially inhibited, preferablyby at least 50% reduction in activity, more preferably by at least 80%reduction in activity, and most preferably by at least 90% reduction inactivity.

[0025] Examples of the tissue or organ transplant include, but are notlimited to, stem cells, bone marrow, heart, liver, kidney, lung,pancreas, small intestine, cornea, and skin. Examples of the ADAinhibitor include, but are not limited to, pentostatin, fludarabinemonophosphate, and cladribine.

[0026] In one embodiment, the transplant is stored in a preservationsolution containing pentostatin in an amount sufficient to inhibitactivity of T-lymphocytes of the transplant. An example of commerciallyavailable preservation solutions is Plegisol (Abbott). The preservationsolution may also contain conventional co-solvents, excipients,stabilizing agents and/or buffering agents.

[0027] In another embodiment, the transplant is washed with a buffercontaining pentostatin prior to storage or transplantation. In this way,the risk of developing acute GVHD upon transplantation should besignificantly reduced, and the host is not only protected from GVHD butalso from potential side effects of pentostatin.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides methods for prevention andclinical treatment of various forms of graft-versus-host disease (GVHD)by using inhibitors of adenosine deaminase (ADA). In particular, novelformulations and dosing regimens of ADA inhibitors such as pentostatinare provided for the treatment in order to specifically suppressT-lymphocyte mediated immune responses while minimizing systemictoxicity of the drug, especially myelosuppression.

[0029] In essence, the methods operate by exploiting the effects ofcertain adenosine analogs such as pentostatin on ADA-dependentT-lymphocytes. It is believed that pentostatin asserts potent inhibitoryeffects on ADA, which ultimately leads to apoptosis of T-lymphocytes(i.e., T-cells). However, physiological functions of B-cells that areT-cell independent and hemopoietic stem cells are largely unaffected. Byexploiting the preferential toxic effects of an ADA inhibitor onT-cells, patients can be treated with the drug prior to or within a timewindow post transplantation to prevent onset of GVHD or organ rejection.Further, patients with acute or chronic GVHD can be treated with thedrug to reduce the pathological symptoms with minimal myelosuppression.In addition, the ADA inhibitor may be used for ex vivo treatment orconditioning of transplants to specifically activate T-lymphocytes fromthe donor without substantially affecting rate of engraftment.

[0030] The following sections describe in detail the mechanisms ofactions of the ADA-inhibitors, their formulations and methods of the usefor prevention and clinical treatment of GVHD, and for ex vivoconditioning of the transplants

[0031] 1. Adenosine Deaminase (ADA) and its Inhibitors

[0032] ADA is a 41 kD protein expressed in all tissues with highestexpression in lymphocytes. ADA participates in the purine metabolismwhere it degrades either adenosine or 2′-deoxyadenosine producinginosine or 2′-deoxyinosine, respectively. It has been found that theactivity of ADA is subject to changes depending upon the degree ofactivity of the cell, i.e. whether differentiation or proliferationoccurs (Trotta, P. P. and Balis M. E. (1977) Cancer Research 37:2297-2305). A genetic deficiency of ADA may cause severe combinedimmunodeficiency. Dighiero, G. (1996) “Adverse and beneficialimmunological effects of purine nucleoside analogues,” Hematol CellTher, 38:575-581.

[0033] Certain adenosine analogs have been found to have inhibitoryeffects on ADA. These compounds include, but are not limited to,pentostatin (2′-deoxycoformycin, also referred to as dCF, or NIPENT®);fludarabine monophosphate (FLU), a fluorinated analogue of adenine thatis relatively resistant to adenosine-deaminase, and cladribine(2-chloro-2′-deoxyadenosine or 2CDA) which is also resistant toadenosine deaminase through introduction of a chlorine at the 2 carbon.These adenosine analogs that inhibits ADA activity and/or cause abnormalaccumulation of deoxyadenosine and adenosine in the cell are hereincollectively referred to as “ADA inhibitors”.

[0034] While the exact nature of the ADA pathway intervention by theseADA inhibitors seems unclear, it may be that these compounds that areresistant to cellular deamination might mimic the ADA-deficient state.Lack of ADA seems to lead to a build up of deoxyadenosine and adenosinetriphosphate in the cell, thus fatally accelerating DNA strand breaks inthe cell. Under normal conditions, cells are continuously breaking andrejoining DNA. When this physiological process is accelerated by theeffect of excess adenosine triphosphate, it leads to consumption of NADfor poly-ADP-ribose synthesis. This polymer is produced fromnicotinamide adenosine dinucleotides (NAD) in a reaction catalyzed bythe chromatin-associated poly(ADP-ribose) synthetase, leading to adepletion of the NAD content of the cell. This depletion induces aprofound alteration of cellular reducing power, because of lethal ADPand ATP depletion.

[0035] The result is programmed cell death through activation of aCa²⁺-, Mg²⁺-dependent endonuclease. Hence, it appears that the ADAinhibitors according to the invention can act on cells, withpreferential lymphocytic activity, via an apoptotic process. The factthat supplementation of a cell medium with the NAD precursor ofnicotinamide or 3-aminobenzamide, an inhibitor of poly (ADP-ribose)synthetase, prevented NAD depletion and reduces 2CDA toxicity, tends tosupport this hypothesis.

[0036] The ADA inhibitors listed above may affect the ADA pathway indifferent manners but ultimately leads accumulation of adenosinetriphosphate and promotes apoptosis of cells, especially lymphocytesthat have low levels of the nucleoside-cleaving enzyme 5′-nucleotidaseare particularly sensitive to these antimetabolic effects. Pentostatin,for example, has been shown to be an irreversible inhibitor of ADA. Byfavoring the predominance of deoxycytidine kinase (DCK) over thedephosphorylating enzyme 5-nucleotidase in lymphocytes it induces apreferential accumulation of deoxyadenosine-5′-triphosphate (dATP). Bycomparison, FLU and 2CDA are rather resistant to the enzyme. Both drugsare initially phosphorylated by DCK and contribute to the accumulationof cellular adenosine triphosphate surrogates. As noted above, theaccumulation of adenosine triphosphate, whether by the presumedpentostatin mechanism, or the FLU or 2CDA mechanism, promotes theapoptotic death of the cell.

[0037] Pentostatin has been widely used as an antimetabolite to treatvarious forms of leukemia in the clinic. C. Dearden, et al.,“Deoxycoformycin in the treatment of mature T-cell leukemias”, Brit J.of Can., 64(5):903-906 (November 1991); J. Seymour et al., “Responseduration and recovery of CD4+ lymphocytes following deoxycoformycin ininterferon-α-resistant hairy cell leukemia: 7-year follow-up”, Leukemia,11, 42-47 (1997); J. Johnston et al., “Induction of Apoptosis in CD4+Prolymphocytic Leukemia by Deoxyadenosine and 2′-Deoxycoformycin”,Leukemia Research, 16:8, 781-788 (1992); E. Copelan et al.,“Pharmacologic Marrow Purging in Murine T Cell Leukemia”, Blood,71(6):1656-1661 (June 1988).

[0038] According to the present invention, it is believed that ADAinhibitors can be used to preferentially targeting T-lymphocytes,including those from the donor. By reducing T-lymphocytes presumablythrough apoptosis caused by the ADA inhibitor, attack of the donorT-lymphocytes on the host's organs may be prevented or significantlycompromised.

[0039] 2. Formulations of ADA Inhibitors

[0040] The present invention provides novel formulations and dosingregimens of ADA inhibitors for treating GVHD. By using the methodologyof the present invention, GVHD may be treated in a more efficacious andconvenient way and with a more favorable myelotoxicity profile. In apreferred embodiment, the ADA inhibitor is delivered orally for thetreatment or prevention of GVHD. Optionally, the ADA inhibitor may bedelivered mucosally or nasally for the treatment or prevention of GVHD.Alternatively, the ADA inhibitor may be delivered parenterally, inparticular, intravenously. Preferably, the ADA inhibitor is delivered ata lower dose than those used in an oncological treatment.

[0041] 1) ADA Inhibitors

[0042] The ADA inhibitor referred within is a composition that has aninhibitory effect on biochemical activity of adenosine deaminase (ADA).Examples of the ADA inhibitor include, but are not limited to,pentostatin, fludarabine monophosphate, and cladribine. Other ADAinhibitor may be adenosine analogs that compete with adenosine forbinding to ADA such as 2′-deoxyadenosine, 3′-deoxyadenosine, anddideoxyadenosine.

[0043] Optionally, the ADA inhibitor is in the form of a therapeuticallyacceptable salt. This salt may be prepared in the conventional manner.Salt formers that may, for example, be used are conventional anions orsalts thereof that are physiologically acceptable in the salt form.Examples thereof are: amino acids such as tyrosine or asparagine,sulfates, phosphates, carboxylic acids, tosylates, nitrates, acetates,and long chain fatty acid derivatives of these.

[0044] Should the ADA inhibitor be used in the form of a salt, the saltformer may also be used in excess, i.e. in an amount greater thanequimolar.

[0045] 2) Oral Formulation of ADA Inhibitors

[0046] In one aspect, the ADA inhibitor is formulated for oraladministration. Currently, pentostatin, for example, that is used in theclinic for treating hairy cell leukemia is formulated for intravenous(IV) administration. There are a few practical limitations associatedwith such a dosage form. For example, IV dosing is expensive. Itrequires a highly trained medical professional to administer the IVdose. The dosing involves expensive equipment and materials.Additionally, IV dosing presents increased possibilities of infection,through use of contaminated equipment or accidental contamination, forexample. This is a special concern in health care settings whereincreased incidences of antibiotic resistant bacteria are being noted.

[0047] An oral dosage form may alleviate most, if not all, of theabove-mentioned problems associated with IV or other parenteral dosageforms. However, it is well recognized in the art that deoxyadenosineanalogs such as pentostatin is highly acid-labile. They are highlysusceptible to acid-catalyzed glycosidic cleavage. Therefore, one ofordinary skill in the art would expect that an orally administeredadenosine analog would be cleaved in the stomach, and rendered inactive.In fact, investigators studying pentostatin, have not considered oraladministration of the drug worth studying because of its known acidlability. Marvin M. Chassin et al. “Enzyme Inhibition Titration Assayfor 2′-deoxycoformycin and its Application to the Study of theRelationship Between Drug Concentration and Tissue Adenosine Deaminasein Dogs and Rats” Biochemical Pharmacology 28:1849-1855 (1979).Likewise, other researchers have reported on the acid lability of2′-deoxycoformycin. L. A. al-Razzak et al. “Chemical Stability ofPentostatin (NSC-218321), a Cytotoxic and Immunosuppressant Agent”,Pharm. Res. 7:452-460 (1990).

[0048] Other ADA inhibitors may be expected to have similar acidlability characteristics. A. Tarasiuk et al. “Stability of2-chloro-2′-deoxyadenosine at Various pH and Temperature” Arch. Immunol.Ther. Exp. (Warsz) 42:13-15 (1994); T. Ono “2′-Fluoro Modified NucleicAcids: Polymerase-directed Synthesis, Properties and Stability toAnalysis by Matrix-Assisted Laser Desorption/Ionization MassSpectrometry” Nucleic Acids Res. 25:4581-4588 (1997).

[0049] Against this mainstream of thought the inventors believe that theADA inhibitors can be formulated for oral administration andconveniently used to treat various forms of GVHD. It is also believedthat it is possible to achieve bioavailability of the ADA inhibitorusing an oral dosage form and the effects of the drug on the patient inan oral dosage form should be reasonably close to those achieved usingan IV dosage form.

[0050] Various oral dosage forms of the ADA inhibitor may be used in thepractice of the invention. For example, the ADA inhibitor may be mixedwith a pharmacologically acceptable liquid and swallowed. The ADAinhibitor also may be compounded into tablets, capsules, pills,lozenges, etc. using conventional compounding techniques.

[0051] In one embodiment, the ADA inhibitor may be administered withvarious agents to reduce acid concentration in the stomach. This reducesacid lability and allows for enhanced concentrations of the ADAinhibitor for enhanced gastric and/or intestinal absorption. Forexample, the adenosine analog may be coadministered with an H2 inhibitorsuch as cimetidine, an acid neutralizer such as calcium carbonate, or aproton pump inhibitor.

[0052] Furthermore, the ADA inhibitor may be (co)administered using adosage form that reduces the effect of acid lability on theirbioavailability. (Co)administration within the context of this inventionmay be taken to mean administration, coadministration, or both.Coadministration in the context of this invention may be defined to meanthe administration of more than one therapeutic in the course of acoordinated treatment to achieve an improved clinical outcome. Suchcoadministration may also be coextensive, that is, occurring duringoverlapping periods of time.

[0053] In one variation, the ADA inhibitor such as pentostatin isformulated in a dosage form that reduces acid lability of the compound,thereby enhancing the bioavailability the compound. For example,pentostatin may be compounded with polymer matrices that are erodiblechemically or biologically.

[0054] Optionally, the ADA inhibitor may be coated with an entericcoating to prevent ready decomposition in the stomach. The entericcoating may comprise hydroxypropyl-methylcellulose phthalate,methacrylic acid-methacrylic acid ester copolymer, polyvinylacetate-phthalate and cellulose acetate phthalate.

[0055] The dosage form of the ADA inhibitor may also be a soliddispersion with a water soluble or a water insoluble carrier. Examplesof water soluble or water insoluble carrier include, but are not limitedto, polyethylene glycol, polyvinylpyrrolidone,hydroxypropylmethylcellulose, phosphatidylcholine, polyoxyethylenehydrogenated castor oil, hydroxypropylmethylcellulose phthalate,carboxymethylethylcellulose, or hydroxypropylmethylcellulose, ethylcellulose, or stearic acid.

[0056] Optionally, oral dosage form of the ADA inhibitor may be acomplex between an ion exchange resin and the ADA inhibitor.

[0057] In another aspect, the ADA inhibitor may be formulated for acontrolled release. Controlled release within the scope of thisinvention can be taken to mean any one of a number of extended releasedosage forms.

[0058] The following terms may be considered to be substantiallyequivalent to controlled release, for the purposes of the presentinvention: continuous release, controlled release, delayed release,depot, gradual release, long-term release, programmed release, prolongedrelease, proportionate release, protracted release, repository, retard,slow release, spaced release, sustained release, time coat, timedrelease, delayed action, extended action, layered-time action, longacting, prolonged action, repeated action, slowing acting, sustainedaction, sustained-action medications, and extended release. Furtherdiscussions of these terms may be found in Lesczek Krowczynski“Extended-Release Dosage Forms”, 1987 (CRC Press, Inc.).

[0059] The various controlled release technologies cover a very broadspectrum of drug dosage forms. Controlled release technologies include,but are not limited to physical systems and chemical systems.

[0060] Physical systems include, but not limited to, reservoir systemswith rate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., non-erodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., non-erodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

[0061] Chemical systems include, but are not limited to, chemicalerosion of polymer matrices (e.g., heterogeneous, or homogeneouserosion), or biological erosion of a polymer matrix (e.g.,heterogeneous, or homogeneous). Additional discussion of categories ofsystems for controlled release may be found in Agis F. Kydonieus,“Controlled Release Technologies: Methods, Theory and Applications” 1980(CRC Press, Inc.).

[0062] Controlled release drug delivery systems may also be categorizedunder their basic technology areas, including, but not limited to,rate-preprogrammed drug delivery systems, activation-modulated drugdelivery systems, feedback-regulated drug delivery systems, andsite-targeting drug delivery systems.

[0063] In rate-preprogrammed drug delivery systems, release of drugmolecules from the delivery systems “preprogrammed” at specific rateprofiles. This may be accomplished by system design, which controls themolecular diffusion of drug molecules in and/or across the barriermedium within or surrounding the delivery system. Fick's laws ofdiffusion are often followed.

[0064] In activation-modulated drug delivery systems, release of drugmolecules from the delivery systems is activated by some physical,chemical or biochemical processes and/or facilitated by the energysupplied externally. The rate of drug release is then controlled byregulating the process applied, or energy input.

[0065] In feedback-regulated drug delivery systems, release of drugmolecules from the delivery systems may be activated by a triggeringevent, such as a biochemical substance, in the body. The rate of drugrelease is then controlled by the concentration of triggering agentdetected by a sensor in the feedback regulated mechanism.

[0066] In a site-targeting controlled-release drug delivery system, thedrug delivery system targets the active molecule to a specific site ortarget tissue or cell. This may be accomplished, for example, by aconjugate including a site specific targeting moiety that leads the drugdelivery system to the vicinity of a target tissue (or cell), asolubilizer that enables the drug delivery system to be transported toand preferentially taken up by a target tissue, and a drug moiety thatis covalently bonded to the polymer backbone through a spacer andcontains a cleavable group that can be cleaved only by a specific enzymeat the target tissue.

[0067] While a preferable mode of controlled release drug delivery willbe oral, other modes of delivery of controlled release compositionsaccording to this invention may be used to treat or prevent GVHD. Theseinclude mucosal delivery, nasal delivery, ocular delivery, transdermaldelivery, parenteral controlled release delivery, vaginal delivery,rectal delivery and intrauterine delivery. All of these dosage forms maybe manufactured using conventional techniques, together with thetechniques discussed herein.

[0068] There are a number of controlled release drug formulations thatare developed preferably for oral administration. These include, but arenot limited to, osmotic pressure-controlled gastrointestinal deliverysystems; hydrodynamic pressure-controlled gastrointestinal deliverysystems; membrane permeation-controlled gastrointestinal deliverysystems, which include microporous membrane permeation-controlledgastrointestinal delivery devices; gastric fluid-resistant intestinetargeted controlled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien “Novel DrugDelivery Systems” 1992 (Marcel Dekker, Inc.). Some of these formulationswill now be discussed in more detail.

[0069] Enteric coatings may be applied to tablets to prevent the releaseof drugs in the stomach either to reduce the risk of unpleasant sideeffects or to maintain the stability of the drug which might otherwisebe subject to degradation due to expose to the gastric environment. Mostpolymers that are used for this purpose are polyacids that function byvirtue of the fact that their solubility in aqueous medium ispH-dependent, and they require conditions with a pH higher than normallyencountered in the stomach.

[0070] Enteric coatings may be used to coat a solid or liquid dosageform of adenosine analogs according to the invention. Enteric coatingspromote the inventive adenosine analogs remaining physicallyincorporated in the dosage form for a specified period when exposed togastric juice. Yet the enteric coatings are designed to disintegrate inintestinal fluid for ready absorption. Delay of the adenosine analogsabsorption is dependent on the rate of transfer through thegastrointestinal tract, and so the rate of gastric emptying is animportant factor. Some investigators have reported that a multiple-unittype dosage form, such as granules, may be superior to a single-unittype. Therefore, in a preferable embodiment, the ADA inhibitor accordingto the invention may be contained in an enterically coated multiple-unitdosage form. In a more preferable embodiment, the dosage form of the ADAinhibitor according to the invention is prepared by spray-coatinggranules of an adenosine analog-enteric coating agent solid dispersionon an inert core material. These granules can result in prolongedabsorption of the drug with good bioavailability.

[0071] Typical enteric coating agents include, but are not limited to,hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylicacid ester copolymer, polyvinyl acetate-phthalate and cellulose acetatephthalate. Akihiko Hasegawa “Application of solid dispersions ofNifedipine with enteric coating agent to prepare a sustained-releasedosage form” Chem. Pharm. Bull. 33: 1615-1619 (1985). Various entericcoating materials may be selected on the basis of testing to achieve anenteric coated dosage form designed ab initio to have a preferablecombination of dissolution time, coating thicknesses and diametralcrushing strength. S. C. Porter et al. “The Properties of Enteric TabletCoatings Made From Polyvinyl Acetate-phthalate and Cellulose acetatePhthalate”, J. Pharm. Pharmacol. 22:42p (1970).

[0072] On occasion, the performance of an enteric coating may hinge onits permeability. S. C. Porter et al., “The Permeability of EntericCoatings and the Dissolution Rates of Coated Tablets”, J. Pharm.Pharmacol. 34: 5-8 (1981). With such oral drug delivery systems, thedrug release process may be initiated by diffusion of aqueous fluidsacross the enteric coating. Investigations have suggested osmoticdriven/rupturing affects as important release mechanisms from entericcoated dosage forms. Roland Bodmeier et al. “Mechanical Properties ofDry and Wet Cellulosic and Acrylic Films Prepared from Aqueous ColloidalPolymer Dispersions used in the Coating of Solid Dosage Forms”Pharmaceutical Research, 11: 882-888 (1994).

[0073] Another type of useful oral controlled release structure is asolid dispersion. A solid dispersion may be defined as a dispersion ofone or more active ingredients in an inert carrier or matrix in thesolid state prepared by the melting (fusion), solvent, ormelting-solvent method. Akihiko Hasegawa “Super Saturation Mechanism ofDrugs from Solid Dispersions with Enteric Coating Agents”, Chem. Pharm.Bull. 36: 4941-4950 (1998). The solid dispersions may be also calledsolid-state dispersions. The term “coprecipitates” may also be used torefer to those preparations obtained by the solvent methods.

[0074] Solid dispersions may be used to improve the solubilities and/ordissolution rates of the ADA inhibitor according to the invention thatmay be poorly water-soluble. Hiroshi Yuasa, et al. “Application of theSolid Dispersion Method to the Controlled Release Medicine. III. Controlof the Release Rate of Slightly Water-Soluble Medicine From SolidDispersion Granules” Chem. Pharm. Bull. 41:397-399 (1993). The soliddispersion method was originally used to enhance the dissolution rate ofslightly water-soluble medicines by dispersing the medicines intowater-soluble carriers such as polyethylene glycol orpolyvinylpyrrolidone, Hiroshi Yuasa, et al. “Application of the SolidDispersion Method to the Controlled Release of Medicine. IV. PreciseControl of the Release Rate of a Water-Soluble Medicine by Using theSolid Dispersion Method Applying the Difference in the Molecular Weightof a Polymer” Chem. Pharm. Bull. 41:933-936 (1993).

[0075] The selection of the carrier may have an influence on thedissolution characteristics of the dispersed drug because thedissolution rate of a component from a surface may be affected by othercomponents in a multiple component mixture. For example, a water-solublecarrier may result in a fast release of the drug from the matrix, or apoorly soluble or insoluble carrier may lead to a slower release of thedrug from the matrix. The solubility of poorly water soluble adenosineanalogs according to the invention may also be increased owing to someinteraction with the carriers.

[0076] Examples of carriers useful in solid dispersions according to theinvention include, but are not limited to, water-soluble polymers suchas polyethylene glycol, polyvinylpyrrolidone, orhydroxypropylmethylcellulose. Akihiko Hasegawa, “Application of SolidDispersions of Nifedipine with Enteric Coating Agent to Prepare aSustained-release Dosage Form” Chem. Pharm. Bull. 33: 1615-1619 (1985).

[0077] Alternate carriers include phosphatidylcholine. Makiko Fuji, etal. “The Properties of Solid Dispersions of Indomethacin, Ketoprofen andFlurbiprofen in Phosphatidylcholine” Chem. Pharm. Bull. 36:2186-2192(1988). Phosphatidylcholine is an amphoteric but water-insoluble lipid,which may improve the solubility of otherwise insoluble adenosineanalogs in an amorphous state in phosphatidylcholine solid dispersions.See Makiko Fuji, et al.,“Dissolution of Bioavailability of Phenytoin inSolid Dispersion with Phosphatidylcholine” Chem. Pharm. Bull36:4908-4913 (1988).

[0078] Other carriers include polyoxyethylene hydrogenated castor oil.Katsuhiko Yano, et al. “In-Vitro Stability and In-Vivo AbsorptionStudies of Colloidal Particles Formed From a Solid Dispersion System”Chem. Pharm. Bull 44:2309-2313 (1996). Poorly water-soluble adenosineanalogs according to the invention may be included in a solid dispersionsystem with an enteric polymer such as hydroxypropylmethylcellulosephthalate and carboxymethylethylcellulose, and a non-enteric polymer,hydroxypropylmethylcellulose. See Toshiya Kai, et al., “Oral AbsorptionImprovement of Poorly Soluble Drug Using Soluble Dispersion Technique”,Chem. Pharm. Bull. 44:568-571 (1996). Another solid dispersion dosageform includes incorporation of the ADA inhibitor with ethyl celluloseand stearic acid in different ratios. Kousuke Nakano, et al. “OralSustained-Release Cisplatin Preparations for Rats and Mice” J. Pharm.Pharmacol. 49:485-490 (1997).

[0079] There are various methods commonly known for preparing soliddispersions. These include, but are not limited to the melting method,the solvent method and the melting-solvent method.

[0080] In the melting method, the physical mixture of a drug in awater-soluble carrier is heated directly until it melts. The meltedmixture is then cooled and solidified rapidly while rigorously stirred.The final solid mass is crushed, pulverized and sieved. Using thismethod a super saturation of a solute or drug in a system can often beobtained by quenching the melt rapidly from a high temperature. Undersuch conditions, the solute molecule may be arrested in solvent matrixby the instantaneous solidification process. A disadvantage is that manysubstances, either drugs or carriers, may decompose or evaporate duringthe fusion process at high temperatures. However, this evaporationproblem may be avoided if the physical mixture is heated in a sealedcontainer. Melting under a vacuum or blanket of an inert gas such asnitrogen may be employed to prevent oxidation of the drug or carrier.

[0081] The solvent method has been used in the preparation of solidsolutions or mixed crystals of organic or inorganic compounds. Solventmethod dispersions may be prepared by dissolving a physical mixture oftwo solid components in a common solvent, followed by evaporation of thesolvent. The main advantage of the solvent method is that thermaldecomposition of drugs or carriers may be prevented because of the lowtemperature required for the evaporation of organic solvents. However,some disadvantages associated with this method are the higher cost ofpreparation, the difficulty in completely removing liquid solvent, thepossible adverse effect of its supposedly negligible amount of thesolvent on the chemical stability of the drug.

[0082] Another method of producing solid dispersions is themelting-solvent method. It is possible to prepare solid dispersions byfirst dissolving a drug in a suitable liquid solvent and thenincorporating the solution directly into a melt of polyethylene glycol,obtainable below 70 degrees, without removing the liquid solvent. Theselected solvent or dissolved adenosine analogs may be selected suchthat the solution is not miscible with the melt of polyethylene glycol.The polymorphic form of the adenosine analogs may then be precipitatedin the melt. Such a unique method possesses the advantages of both themelting and solvent methods. Win Loung Chiou, et al. “PharmaceuticalApplications of Solid Dispersion Systems” J. Pharm. Sci. 60:1281-1301(1971).

[0083] Another controlled release dosage form is a complex between anion exchange resin and the ADA inhibitor according to the invention. Ionexchange resin-drug complexes have been used to formulatesustained-release products of acidic and basic drugs. In one preferableembodiment, a polymeric film coating is provided to the ion exchangeresin-drug complex particles, making drug release from these particlesdiffusion controlled. See Y. Raghunathan et al. “Sustained-released drugdelivery system I: Coded ion-exchange resin systems forphenylpropanolamine and other drugs” J. Pharm. Sciences 70: 379-384(1981).

[0084] Injectable micro spheres are another controlled release dosageform. Injectable micro spheres may be prepared by non-aqueous phaseseparation techniques, and spray-drying techniques. Micro spheres may beprepared using polylactic acid or copoly(lactic/glycolic acid).Shigeyuki Takada “Utilization of an Amorphous Form of a Water-SolubleGPIIb/IIIa Antagonist for Controlled Release From Biodegradable Microspheres” Pharm. Res. 14:1146-1150 (1997), and ethyl cellulose, YoshiyukiKoida “Studies on Dissolution Mechanism of Drugs from Ethyl CelluloseMicrocapsules” Chem. Pharm. Bull. 35:1538-1545 (1987).

[0085] Other controlled release technologies that may be used in thepractice of this invention are quite varied. They include SODAS, INDAS,IPDAS, MODAS, EFVAS, PRODAS, and DUREDAS. SODAS are multi particulatedosage forms utilizing controlled release beads. INDAS are a family ofdrug delivery technologies designed to increase the solubility of poorlysoluble drugs. IPDAS are multi particulate tablet formation utilizing acombination of high density controlled release beads and an immediaterelease granulate. MODAS are controlled release single unit dosageforms. Each tablet consists of an inner core surrounded by asemipermeable multiparous membrane that controls the rate of drugrelease. EFVAS is an effervescent drug absorption system. PRODAS is afamily of multi particulate formulations utilizing combinations ofimmediate release and controlled release mini-tablets. DUREDAS is abilayer tablet formulation providing dual release rates within the onedosage form. Although these dosage forms are known to one of skill,certain of these dosage forms will now be discussed in more detail.

[0086] INDAS was developed specifically to improve the solubility andabsorption characteristics of poorly water soluble drugs. Solubilityand, in particular, dissolution within the fluids of thegastrointestinal tract is a key factor in determining the overall oralbioavailability of poorly water soluble drug. By enhancing solubility,one can increase the overall bioavailability of a drug with resultingreductions in dosage. INDAS takes the form of a high energy matrixtablet. In a preferred embodiment of the invention production involvesincluding adenosine analogs in an amorphous form together with acombination of energy, excipients, and unique processing procedures.

[0087] Once included in the desirable physical form, the resultant highenergy complex may be stabilized by an absorption process that utilizesa novel polymer cross-linked technology to prevent recrystallization.The combination of the change in the physical state of the ADA inhibitoraccording to the invention coupled with the solubilizing characteristicsof the excipients employed enhances the solubility of the ADA inhibitoraccording to the invention. The resulting absorbed amorphous drugcomplex granulate may be formulated with a gel-forming erodable tabletsystem to promote substantially smooth and continuous absorption.

[0088] IPDAS is a multi-particulate tablet technology that may enhancethe gastrointestinal tolerability of potential irritant and ulcerogenicdrugs. Intestinal protection is facilitated by the multi-particulatenature of the IPDAS formulation which promotes dispersion of an irritantADA inhibitor according to the invention throughout the gastrointestinaltract. Controlled release characteristics of the individual beads mayavoid high concentration of drug being both released locally andabsorbed systemically. The combination of both approaches serves tominimize the potential harm of the ADA inhibitor according to theinvention with resultant benefits to patients.

[0089] IPDAS is composed of numerous high density controlled releasebeads. Each bead may be manufactured by a two step process that involvesthe initial production of a micromatrix with embedded adenosine analogsaccording to the invention and the subsequent coating of thismicromatrix with polymer solutions that form a rate limitingsemipermeable membrane in vivo. Once an IPDAS tablet is ingested, it maydisintegrate and liberate the beads in the stomach. These beads maysubsequently pass into the duodenum and along the gastrointestinaltract, preferably in a controlled and gradual manner, independent of thefeeding state. The release of the ADA inhibitor occurs by diffusionprocess through the micromatrix and subsequently through the pores inthe rate controlling semipermeable membrane. The release rate from theIPDAS tablet may be customized to deliver a drug-specific absorptionprofile associated with optimized clinical benefit. Should a fast onsetof activity be necessary, immediate release granulate may be included inthe tablet. The tablet may be broken prior to administration, withoutsubstantially compromising drug release, if a reduced dose is requiredfor individual titration.

[0090] MODAS is a drug delivery system that may be used to control theabsorption of a water soluble ADA inhibitor according to the invention.Physically MODAS is a non-disintegrating table formulation thatmanipulates drug release by a process of rate limiting diffusion by asemipermeable membrane formed in vivo. The diffusion process essentiallydictates the rate of presentation of drug to the gastrointestinalfluids, such that the uptake into the body is controlled. Because of theminimal use of excipients, MODAS can readily accommodate small dosagesize forms. Each MODAS tablet begins as a core containing active drugplus excipients. This core is coated with a solution of insolublepolymers and soluble excipients. Once the tablet is ingested, the fluidof the gastrointestinal tract may dissolve the soluble excipients in theouter coating leaving substantially the insoluble polymer. What resultsis a network of tiny, narrow channels connecting fluid from thegastrointestinal tract to the inner drug core of water soluble drug.This fluid passes through these channels, into the core, dissolving thedrug, and the resultant solution of drug may diffuse out in a controlledmanner. This may permit both controlled dissolution and absorption. Anadvantage of this system is that the drug releasing pores of the tabletare distributed over substantially the entire surface of the tablet.This facilitates uniform drug absorption and reduces aggressiveunidirectional drug delivery. MODAS represents a very flexible dosageform in that both the inner core and the outer semipermeable membranemay be altered to suit the individual delivery requirements of a drug.In particular, the addition of excipients to the inner core may help toproduce a micro-environment within the tablet that facilitates morepredictable release and absorption rates. The addition of an immediaterelease outer coating may allow for development of combination products.

[0091] Additionally, PRODAS may be used to deliver the ADA inhibitoraccording to the invention. PRODAS is a multi particulate drug deliverytechnology based on the production of controlled release mini tablets inthe size range of 1.5 to 4 mm in diameter. The PRODAS technology is ahybrid of multi particulate and hydrophilic matrix tablet approaches,and may incorporate, in one dosage form, the benefits of both these drugdelivery systems.

[0092] In its most basic form, PRODAS involves the direct compression ofan immediate release granulate to produce individual mini tablets thatcontain adenosine analogs according to the invention. These mini tabletsare subsequently incorporated into hard gels and capsules that representthe final dosage form. A more beneficial use of this technology is inthe production of controlled release formulations. In this case, theincorporation of various polymer combinations within the granulate maydelay the release rate of drugs from each of the individual minitablets. These mini tablets may subsequently be coated with controlledrelease polymer solutions to provide additional delayed releaseproperties. The additional coating may be necessary in the case ofhighly water soluble drugs or drugs that are perhaps gastro-irritantswhere release can be delayed until the formulation reaches more distalregions of the gastrointestinal tract. One value of PRODAS technologylies in the inherent flexibility to formulation whereby combinations ofmini tablets, each with different release rates, are incorporated intoone dosage form. As well as potentially permitting controlled absorptionover a specific period, this also may permit targeted delivery of drugto specific sites of absorption throughout the gastrointestinal tract.Combination products also may be possible using mini tablets formulatedwith different active ingredients.

[0093] DUREDAS is a bilayer tableting technology that may be used in thepractice of the invention. DUREDAS was developed to provide for twodifferent release rates, or dual release of a drug from one dosage form.The term bilayer refers to two separate direct compression events thattake place during the tableting process. In a preferable embodiment, animmediate release granulate is first compressed, being followed by theaddition of a controlled release element which is then compressed ontothis initial tablet. This may give rise to the characteristic bilayerseen in the final dosage form.

[0094] The controlled release properties may be provided by acombination of hydrophilic polymers. In certain cases, a rapid releaseof the ADA inhibitor according to the invention may be desirable inorder to facilitate a fast onset of therapeutic affect. Hence one layerof the tablet may be formulated as an immediate release granulate. Bycontrast, the second layer of the tablet may release the drug in acontrolled manner, preferably through the use of hydrophilic polymers.This controlled release may result from a combination of diffusion anderosion through the hydrophilic polymer matrix.

[0095] A further extension of DUREDAS technology is the production ofcontrolled release combination dosage forms. In this instance, twodifferent ADA inhibitors according to the invention may be incorporatedinto the bilayer tablet and the release of drug from each layercontrolled to maximize therapeutic affect of the combination.

[0096] One preferable example of coadministration is the combination ofdeoxyadenosines with pentostatin. This combination may operatesynergistically, to obtain a differential effect over either of thetherapeutic agents administered separately. It has been reported thatpentostatin enhances the clinical anti-HIV activity of related adenosineanalogs presumably due to prevention of degradation of the adenosineanalogs by adenosine deaminase. G. S. Ahluwalia, et al., “Enhancement by2′-deoxycoformycin of the 5″-Phosphorylation and Anti-Humanimmunodeficiency virus activity of 2′3′-dideoxyadenosine and2′-beta-fluor-2′, 3′-dideoxyadenosine”, Molec. Pharmacol. 46:1002-1008(1994).

[0097] Furthermore, the ADA inhibitor may be administered orcoadministered with conventional pharmaceutical excipients andadditives. These include, but are not limited to, gelatin, naturalsugars such as raw sugar or lactose, lecithin, pectin, starches (forexample corn starch or amylose), dextran, polyvinyl pyrrolidone,polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium,silica gel (for example colloidal), cellulose, cellulose derivatives(for example cellulose ethers in which the cellulose hydroxy groups arepartially etherified with lower saturated aliphatic alcohols and/orlower saturated, aliphatic oxyalcohols, for example methyl oxypropylcellulose, methyl cellulose, hydroxypropyl methyl cellulose,hydroxypropyl methyl cellulose phthalate), fatty acids as well asmagnesium, calcium or aluminum salts of fatty acids with 12 to 22 carbonatoms, in particular saturated (for example stearates), emulsifiers,oils and fats, in particular vegetable (for example, peanut oil, castoroil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil,sunflower seed oil, cod liver oil, in each case also optionallyhydrated); glycerol esters and polyglycerol esters of saturated fattyacids C₁₂H₂₄O₂ to C₁₈H₃₆O₂ and their mixtures, it being possible for theglycerol hydroxy groups to be totally or also only partly esterified(for example mono-, di- and triglycerides); pharmaceutically acceptablemono- or multivalent alcohols and polyglycols such as polyethyleneglycol and derivatives thereof, esters of aliphatic saturated orunsaturated fatty acids (2 to 22 carbon atoms, in particular 10-18carbon atoms) with monovalent aliphatic alcohols (1 to 20 carbon atoms)or multivalent alcohols such as glycols, glycerol, diethylene glycol,pentacrythritol, sorbitol, mannitol and the like, which may optionallyalso be etherified, esters of citric acid with primary alcohols, aceticacid, urea, benzyl benzoate, dioxolanes, glyceroformals,tetrahydrofurfuryl alcohol, polyglycol ethers with C₁-C₁₂-alcohols,dimethylacetamide, lactamides, lactates, ethylcarbonates, silicones (inparticular medium-viscous polydimethyl siloxanes), calcium carbonate,sodium carbonate, calcium phosphate, sodium phosphate, magnesiumcarbonate and the like.

[0098] Other auxiliary substances that may be used are those which causedisintegration (so-called disintegrants), such as: cross-linkedpolyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethylcellulose or microcrystalline cellulose. Conventional coating substancesmay also be used to produce the oral dosage form. Those that may forexample be considered are: polymerizates as well as copolymerizates ofacrylic acid and/or methacrylic acid and/or their esters;copolymerizates of acrylic and methacrylic acid esters with a lowerammonium group content (for example EudragitR RS), copolymerizates ofacrylic and methacrylic acid esters and trimethyl ammonium methacrylate(for example EudragitR RL); polyvinyl acetate; fats, oils, waxes, fattyalcohols; hydroxypropyl methyl cellulose phthalate or acetate succinate;cellulose acetate phthalate, starch acetate phthalate as well aspolyvinyl acetate phthalate, carboxy methyl cellulose; methyl cellulosephthalate, methyl cellulose succinate, -phthalate succinate as well asmethyl cellulose phthalic acid half ester; zein; ethyl cellulose as wellas ethyl cellulose succinate; shellac, gluten; ethylcarboxyethylcellulose; ethacrylate-maleic acid anhydride copolymer; maleic acidanhydride-vinyl methyl ether copolymer; styrol-maleic acidcopolymerizate; 2-ethyl-hexyl-acrylate maleic acid anhydride; crotonicacid-vinyl acetate copolymer; glutaminic acid/glutamic acid estercopolymer; carboxymethylethylcellulose glycerol monooctanoate; celluloseacetate succinate; polyarginine.

[0099] Plasticizing agents that may be considered as coating substancesare: citric and tartaric acid esters (acetyl-triethyl citrate, acetyltributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters(glycerol diacetate, -triacetate, acetylated monoglycerides, castoroil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-,dipropylphthalate), di-(2-methoxy- or 2-ethoxyethyl)-phthalate,ethylphthalyl glycolate, butylphthalylethyl glycolate andbutylglycolate; alcohols (propylene glycol, polyethylene glycol ofvarious chain lengths), adipates (diethyladipate, di-(2-methoxy- or2-ethoxyethyl)-adipate; benzophenone; diethyl- and diburylsebacate,dibutylsuccinate, dibutyltartrate; diethylene glycol dipropionate;ethyleneglycol diacetate, -dibutyrate, -dipropionate; tributylphosphate, tributyrin; polyethylene glycol sorbitan monooleate(polysorbates such as Polysorbar 50); sorbitan monooleate.

[0100] As mentioned above, the ADA inhibitor may be orally administeredor coadministered in a liquid dosage form. For the preparation ofsolutions or suspensions it is, for example, possible to use water,particularly sterile water, or physiologically acceptable organicsolvents, such as alcohols (ethanol, propanol, isopropanol,1,2-propylene glycol, polyglycols and their derivatives, fatty alcohols,partial esters of glycerol), oils (for example peanut oil, olive oil,sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovinehoof oil), paraffins, dimethyl sulphoxide, triglycerides and the like.

[0101] In the case of drinkable solutions the following substances maybe used as stabilizers or solubilizers: lower aliphatic mono- andmultivalent alcohols with 2-4 carbon atoms, such as ethanol, n-propanol,glycerol, polyethylene glycols with molecular weights between 200-600(for example 1 to 40% aqueous solution), diethylene glycol monoethylether, 1,2-propylene glycol, organic amides, for example amides ofaliphatic C₁-C₆-carboxylic acids with ammonia or primary, secondary ortertiary C₁-C₄-amines or C₁-C₄-hydroxy amines such as urea, urethane,acetamide, N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethylacetamide, lower aliphatic amines and diamines with 2-6 carbon atoms,such as ethylene diamine, hydroxyethyl theophylline, tromethamine (forexample as 0.1 to 20% aqueous solution), aliphatic amino acids.

[0102] In preparing the inventive compositions, it is possible to useknown and conventional solubilizers or emulsifiers. Solubilizers andemulsifiers that may for example be used are: polyvinyl pyrrolidone,sorbitan fatty acid esters such as sorbitan trioleate, phosphatides suchas lecithin, acacia, tragacanth, polyoxyethylated sorbitan monooleateand other ethoxylated fatty acid esters of sorbitan, polyoxyethylatedfats, polyoxyethylated oleotriglycerides, linolizated oleotriglycerides,polyethylene oxide condensation products of fatty alcohols, alkylphenolsor fatty acids or also 1-methyl-3-(2-hydroxyethyl)imidazolidone-(2). Inthis context, polyoxyethylated means that the substances in questioncontain polyoxyethylene chains, the degree of polymerization of whichgenerally lies between 2 and 40 and in particular between 10 and 20.

[0103] Polyoxyethylated substances of this kind may for example beobtained by reaction of hydroxyl group-containing compounds (for examplemono- or diglycerides or unsaturated compounds such as those containingoleic acid radicals) with ethylene oxide (for example 40 Mol ethyleneoxide per 1 Mol glyceride).

[0104] Examples of oleotriglycerides are olive oil, peanut oil, castoroil, sesame oil, cottonseed oil, corn oil. See also Dr. H. P. Fiedler“Lexikon der Hillsstoffe für Pharmazie, Kostnetik und angrenzendeGebiete” 1971, pages 191-195.

[0105] It is also possible to add preservatives, stabilizers, buffersubstances, flavor correcting agents, sweeteners, colorants,antioxidants and complex formers and the like. Complex formers which maybe for example be considered are: chelate formers such as ethylenediamine retrascetic acid, nitrilotriacetic acid, diethylene triaminepentacetic acid and their salts.

[0106] It may optionally be necessary to stabilize the ADA inhibitorwith physiologically acceptable bases or buffers to a pH range ofapproximately 6 to 9. Preference may be given to as neutral or weaklybasic a pH value as possible (up to pH 8).

[0107] In some dosage forms, it may be useful to include antioxidants orpreservatives. Antioxidants that may for example be used are sodiumsulphite, sodium hydrogen sulphite, sodium metabisulphite, ascorbicacid, ascorbylpalmitate, -myristate, -stearate, gallic acid, gallic acidalkyl ester, butylhydroxyamisol, nordihydroguaiaretic acid, tocopherolsas well as synergists (substances which bind heavy metals throughcomplex formation, for example lecithin, ascorbic acid, phosphoric acidethylene diamine tetracetic acid, citrates, tartrates). Addition ofsynergists substantially increases the antioxygenic effect of theantioxidants.

[0108] Preservatives that may for example be considered are sorbic acid,p-hydroxybenzoic acid esters (for example lower alkyl esters), benzoicacid, sodium benzoate, trichloroisobutyl alcohol, phenol, cresol,benzethonium chloride, chlorhexidine and formalin derivatives.

[0109] The various oral dosage forms can be prepared according toconventional procedures. For example, tablets can be prepared accordingto common tableting procedures. Capsules can be prepared according toconventional encapsulating procedures. Liquid dosage forms may besupplied as a made-up vial, or may be supplied in a lyophilized statefor dilution just prior to administration. Controlled release dosageforms may be prepared according to the particular dosage form beingused, as is discussed briefly above.

[0110] 3) Parenteral Formulation

[0111] Alternatively, the ADA inhibitor such as pentostatin may beformulated for parenteral administration such as intravenous infusion.In a preferred embodiment, pentostatin is parenterally administered tothe patient at a lower dose than the standard dose used for treatinghair cell leukemia (4 mg/m²). Thus, pentostatin may be supplied as asterile, apyrogenic, lyopholized powder in single dose vials at about1-5 mg/vial and more preferably at 2-4 mg/vial. The vials may alsocontain excipients such as mannitol. Pentostatin contained in the singledose vial can be reconstituted by using sterile water, saline or otherinfusion fluid prior to infusion. The pH of the final infusion fluidcontaining low dose pentostatin is preferably maintained between 7.0 and8.5 by addition of sodium hydroxide or hydrochloric acid.

[0112] 3. Method of Use and Dosing Regimen

[0113] The ADA inhibitor may be used to treat patients with variousforms of GVHD including acute and chronic GVHD that is either naive orrefractory to conventional immunosuppressive agents such as steroids andcyclosporin A. The ADA inhibitor may also be used as prophylaxis toprevent onset of GVHD by pretreating the transplant recipient prior tothe transplantation and/or treating the recipient within a certain timewindow post transplantation.

[0114] 1) Treatment of GVHD

[0115] In one embodiment, a method is provided for treating a patientsuffering from GVHD. The method comprises administering to the GVHDpatient a composition including an ADA inhibitor.

[0116] Dosage amounts and frequency will vary according to theparticular ADA inhibitor, the dosage form, and individual patientcharacteristics. Generally speaking, determining the dosage amount andfrequency for a particular ADA inhibitor (e.g., pentostatin), dosageform, and individual patient characteristic can be accomplished usingconventional dosing studies, coupled with appropriate diagnostics. Inpreferable embodiments, the dosage frequency ranges from daily tomonthly doses, more preferably biweekly doses. In other preferableembodiments, the dosage amount ranges from about 0.02 mg/m² to about 20mg/m², more preferably about 0.05 mg/m² to about 5 mg/m², and mostpreferably 0.1 mg/m² to about 1 mg/m².

[0117] A dosage amount below 1 mg/m² is considered to be much lower thanthe standard dose (4 mg/m² once every 2 weeks) used for the treatment ofhairy cell leukemia. According to the present invention, lowering thedosage amount of pentostatin should minimize myelosuppression associatedwith high dose of this antimetabolic drug and yet can still specificallysuppress the GVHD-causing T-lymphocyte mediated immune response.

[0118] In a particular embodiment, an ADA inhibitor such as pentostatinis used to treat patients that have acute Graft vs Host Disease (aGVHD)but failed at least one immunosuppressive regimen such as a regimenincluding steroids such as prednisone and methylprednisolone,cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine, anddaclizumab. For example, hematopoietic stem cell transplant (HSCT)patients manifesting grade 2 or greater aGVHD, who have failed torespond to treatment with at least 2 mg/Kg of methylprednisolone orequivalent corticosteroid or other salvage therapy, can be treated withpentostatin.

[0119] In a preferred embodiment, the HSCT patient withsteroid-refractory aGVHD is treated with pentostatin according to thefollowing plan. The patient is treated with 0.25-1 mg/m²/day as a 20minute intravenous (IV) infusion on days 1, 2 and 3. The patient'sresponse to the treatment is monitored by following for improvement inthe skin, mouth, fascia, and liver. The same dose may be repeated after14 days if there was response to the first 3 days of treatment that wasincomplete or that recurred after a full or partial response.

[0120] Pentostatin may also be administered orally to achieve similarplasma levels as those via IV route of administration. However, foraGVHD patients with severely damaged gastrointestinal (GI) tract IVinfusion may be the preferred route of administration.

[0121] In another embodiment, an ADA inhibitor such as pentostatin isused to treat steroid-refractory chronic Graft vs Host Disease (cGVHD).For example, recipients of allogeneic HSCT developing cGVHD who havefailed to respond to treatment with at least 2 mg/Kg ofmethylprednisolone or equivalent corticosteroid or other salvagetherapy, can be treated with pentostatin via IV or oral administration.

[0122] In a variation of this embodiment, pentostatin is orallyadministrated to the cGVHD patient at a dose between about 1-10 mg/m²,preferably between about 2-6 mg/m², and more preferably between about2-4 mg/m² each day for 3 consecutive days each month. For an averageadult, a dose of 4 mg/m² would be approximately 6-8 mg/day (one and ahalf or two 4 mg tablets or capsules).

[0123] 2) GVHD Prophylaxis

[0124] An ADA inhibitor such as pentostatin can also be used as aprophylaxis to prevent onset of GVHD or to reduce the effects of GVHD.

[0125] Pentostatin may be administered as a GVHD prophylaxisparenterally or orally to a transplant recipient within a predeterminedtime window before or after the transplantation.

[0126] In one embodiment, pentostatin may be administered orally to therecipient on days −3 or −2 (i.e., 3 or 2 days before thetransplantation) as part of a non-myeloablative conditioning regimen,then followed by transplantation such as hematopoietic stem cellinfusion. Alternatively, pentostatin may be administered to therecipient by IV infusion at a dose lower than 2 mg/m², preferably at adose lower than 1 mg/m².

[0127] In myeloablative conditioning regimen, the transplant recipientmay be treated with pentostatin p.o. (i.e., via oral administration) at0.5-2.0 mg/m² on days −14, −13, −12 and −3, −2, −1 with high dosecyclophosphamide and/or busulfan and/or melphalan and/or 1200-1800 cGyirradiation prior to stem cell infusion. Post transplantation therecipient may be continuously treated with pentostatin, on days +8 and+15 preferably iv at a lower dose such as 1-2 mg/m² since patientsreceiving myeloablative conditioning might not be able to tolerate anoral dosage form on days +8 and +15 due to severe mucositis.

[0128] In another embodiment, pentostatin may be administered as a GVHDprophylaxis to a transplant recipient after the transplantation. Forexample, for standard (i.e., myeloablative) transplant ornon-myeloablative stem cell transplant (NST) where pentostatin is notused in the conditioning regimen, pentostatin is administered to thetransplant recipient at 0.5-1.5 mg/m²/day on days +8, +15, +22 and +30following stem cell infusion.

[0129] 4. Combination Therapy for GVHD

[0130] Besides use in a single-agent treatment or prevention of GVHD,the ADA inhibitor such as pentostatin can also be used in a combinationtherapy for acute or chronic GVHD. The combination therapy may havesynergistic therapeutic effects on the patients and thus requires loweramount of pentostatin and the other agent used in conjunction to achievesatisfactory therapeutic efficacy. As a result, potential side effectsassociated with high dose of drugs, such as myelosuppression, arereduced and the patient's quality of life is improved.

[0131] Various other therapeutic agents may be combined with pentostatinfor the treatment or prevention of GVHD. The other therapeutic agentsinclude, but are not limited to, immunosuppressive agents such assteroids (e.g., prednisone and methylprednisolone), cyclophosphamide,cyclosporin A, FK506, thalidomide, azathioprine, monoclonal antibodies(e.g., Daclizumab (anti-interleukin (IL)-2), Infliximab (anti-tumornecrosis factor), MEDI-205 (anti-CD2), abx-cbl (anti-CD147)), andpolyclonal antibodies (e.g., ATG (anti-thymocyte globulin)).

[0132] For example, pentostatin may be combined with a steroid such asmethylprednisolone to treat aGVHD. However, such a combination may betoo broadly immunosuppressive to render the patient more susceptible toopportunistic infection.

[0133] For the treatment of acute GVHD, pentostatin may preferably becombined with monoclonal antibodies which specifically target T-cellssuch as Infliximab, Daclizumab, MEDI-205, or abx-cbl. The monoclonalantibody may be administered at the FDA-approved dosage and by itsstandard route of administration (e.g., IV), followed by oral orparenteral administration of pentostatin as described in detail inSection 3 above.

[0134] For the treatment of chronic GVHD, pentostatin may be combinedwith thalidomide. Pentostatin may also be used in conjunction with otherimmunosuppressive agents as prophylaxis for GVHD post transplantation.For example, the recipient of bone marrow transplant may be treated withpentostatin in conjunction with a standard post infusion regimenincluding mini-methotrexate at 5 mg/m² (as opposed to the conventionaldose at 10-15 mg/m²), cyclosporine A (5-6 mg/Kg/d IV or 10-18 mg/Kg/dorally) and FK506 (0.05-0.1 mg/Kg/d IV or 0.15-0.3 mg/Kg/d orally).

[0135] In addition, pentostatin may be used in conjunction with othertypes of therapy as prophylaxis for GVHD prior to transplantation. Forexample, the recipient of bone marrow transplant may be pretreated withpentostatin in conjunction with TBI (radiation), phototherapy,melphalan, cyclophosphamide or ATG to prevent the onset of GVHD.

[0136] 5. Ex Vivo Treatment of Transplants Using ADA Inhibitors

[0137] In yet another aspect, the invention relates to a method of exvivo or in vitro treatment of blood derived cells, bone marrowtransplants, or other organ transplants. The method comprises treatingthe blood derived cells, bone marrow transplants, or other organtransplants with an ADA inhibitor (e.g., pentostatin) in an effectiveamount such that activities of T-lymphocytes therein are substantiallyinhibited, preferably by at least 50% reduction in activity, morepreferably by at least 80% reduction in activity, and most preferably byat least 90% reduction in activity.

[0138] The invention is practiced in an in vitro or ex vivo environment.All of the discussion above regarding clinical treatment or preventionof GVHD that is relevant to an in vitro or ex vivo environment appliesto this practice. In a particular embodiment, practice of an in vitro orex vivo embodiment of the invention might be useful in the practice ofimmune system transplants, such as bone marrow transplants or peripheralstem cell procurement. In such procedures, the ADA inhibitor might beused, as generally described above, to treat the transplant material toinactivate T-lymphocytes therein so that the T-lymphocyte mediatedimmune response is suppressed upon transplantation.

[0139] For example, the ADA inhibitor may be added to a preservationsolution for an organ transplant in an amount sufficient to inhibitactivity of T-lymphocytes of the organ. Such a preservation solution maybe suitable for preservation of different kind of organs such as heart,kidney and liver as well as tissue therefrom. An example of commerciallyavailable preservation solutions is Plegisol (Abbott), and otherpreservation solutions named in respect of its origins include theUW-solution (University of Wisconsin), the Stanford solution and theModified Collins solution (J. Heart Transplant (1988) Vol.7(6):456-4467). The preservation solution may also contain conventionalco-solvents, excipients, stabilizing agents and/or buffering agents.

[0140] The dosage form of the ADA inhibitor may be a liquid solutionready for use or intended for dilution with a preservation solution.Alternatively, the dosage form may be lyophilized or power filled priorto reconstitution with a preservation solution. The lyophilizedsubstance may contain, if suitable, conventional excipients.

[0141] The preservation solution or buffer containing an ADA inhibitor(e.g., pentostatin) may also be used to wash or rinse an organtransplant prior to transplantation or storage. For example, apreservation solution containing pentostatin may be used to flushperfuse an isolated heart which is then stored at 4° C. in thepreservation solution.

[0142] In another embodiment, practice of the invention might be used tocondition organ transplants prior to transplantation. Prior totransplantation pentostatin may be added to the washing buffer to ridthe transplant of active T-lymphocytes. In this way, the risk ofdeveloping acute GVHD upon transplantation should be significantlyreduced, and the host is not only protected from GVHD but also frompotential side effects of pentostatin.

[0143] The concentration of the ADA inhibitor in the preservationsolution or wash buffer may vary according to the type of transplant.For example, pentostatin at 1 μM may be used to treat an isolated heartprior to transplantation.

[0144] Other applications in vitro or ex vivo using an ADA inhibitorwill occur to one of skill in the art and are therefore contemplated asbeing within the scope of the invention.

What is claimed is:
 1. A method for treating a patient havinggraft-versus-host disease, comprises: administering to the patient anadenosine deaminase inhibitor in a pharmaceutically effective amount. 2.The method of claim 1, wherein the adenosine deaminase inhibitor isselected from the group consisting of pentostatin, fludarabinemonophosphate, and cladribine.
 3. The method of claim 1, wherein theadenosine deaminase inhibitor is administered orally to the patient. 4.The method of claim 1, wherein the adenosine deaminase inhibitor isadministered parenterally to the patient.
 5. The method of claim 1,wherein the patient has acute graft-versus-host disease.
 6. The methodof claim 5, wherein the patient has also failed at least oneimmunosuppressive regimen selected from the group consisting ofprednisone, methylprednisolone, cyclophosphamide, cyclosporin A, FK506,thalidomide, azathioprine, and daclizumab.
 7. The method of claim 5,wherein the patient has received hematopoietic stem cell transplant andmanifests grade 2 or greater acute graft-versus-host disease.
 8. Themethod of claim 7, wherein the adenosine deaminase inhibitor ispentostatin.
 9. The method of claim 8, wherein pentostatin isadministered at 0.25-1 mg/m²/day as a 20 minute intravenous (IV)infusion on days 1, 2 and
 3. 10. The method of claim 5, furthercomprising: monitoring the improvement of the GVHD symptoms in the skin,mouth, fascia, and liver.
 11. The method of claim 11, furthercomprising: repeating the treatment with pentostatin at least once. 12.The method of claim 1, wherein the patient has chronic graft vs hostdisease.
 13. The method of claim 12, wherein the patient has also failedat least one immunosuppressive regimen selected from the groupconsisting of prednisone, methylprednisolone, cyclophosphamide,cyclosporin A, FK506, thalidomide, azathioprine, and daclizumab.
 14. Themethod of claim 11, wherein the patient has received hematopoietic stemcell transplant and has also failed to respond to treatment with atleast 2 mg/Kg of methylprednisolone or equivalent corticosteroid. 15.The method of claim 14, wherein the adenosine deaminase inhibitor ispentostatin.
 16. The method of claim 14, wherein pentostatin isadministered to the patient at a dose between about 1-10 mg/m² each dayfor 3 consecutive days each month.
 17. The method of claim 14, whereinpentostatin is administered to the patient at a dose between about 2-6mg/m² each day for 3 consecutive days each month.
 18. The method ofclaim 14, wherein pentostatin is administered to the patient at a dosebetween about 2-4 mg/m² each day for 3 consecutive days each month. 19.A method for preventing or reducing the risk of developinggraft-versus-host disease in a recipient of an organ or tissuetransplant, comprising: administering to the transplant recipient anadenosine deaminase inhibitor in a pharmaceutically effective amountwithin a predetermined time window before or after the transplantation.20. The method of claim 19, wherein the adenosine deaminase inhibitorinhibitor is selected from the group consisting of pentostatin,fludarabine monophosphate, and cladribine.
 21. The method of claim 19,wherein the adenosine deaminase inhibitor is administered orally to thetransplant recipient.
 22. The method of claim 19, wherein the adenosinedeaminase inhibitor is administered parenterally to the transplantrecipient.
 23. The method of claim 19, wherein the adenosine deaminaseinhibitor is pentostatin.
 24. The method of claim 23, whereinpentostatin is administered orally to the transplant recipient 3 or 2days before the transplantation.
 25. The method of claim 23, whereinpentostatin is administered to the transplant recipient by intravenousinfusion at a dose between about 0.1-2 mg/m².
 26. The method of claim23, wherein pentostatin is administered to the transplant recipient byintravenous infusion at a dose between about 0.5-1 mg/m².
 27. The methodof claim 23, wherein the transplant patient is transplanted withhematopoietic stem cells and treated in a myeloablative conditioningregimen.
 28. The method of claim 27, wherein the transplant recipient istreated with pentostatin via oral administration at about 0.5-2 mg/m² ondays −14, −13, −12 and −3, −2, −1 with 1200-1800 cGy irradiation priorto stem cell infusion.
 29. The method of claim 28, further comprising:intravenously administering to the transplant recipient pentostatin ondays +8 and +15 at a dose between about 1-2 mg/m².
 30. The method ofclaim 23, wherein pentostatin is administered to the transplantrecipient after the transplantation.
 31. The method of claim 30, whereinthe transplant recipient is transplanted with hematopoietic stem cells.32. The method of claim 31, wherein pentostatin is administered to thetransplant recipient at 0.5-1.5 mg/m²/day on days +8, +15, +22 and +30following stem cell infusion.
 33. The method of claim 19, furthercomprising: administering to transplant recipient an immunosuppressiveagent selected from the group consisting of prednisone,methylprednisolone, cyclophosphamide, cyclosporin A, FK506, thalidomide,azathioprine, Daclizumab, Infliximab, MEDI-205, abx-cbl and ATG.
 34. Amethod for ex vivo treatment of a tissue or organ transplant,comprising: treating the tissue or organ transplant with an adenosinedeaminase inhibitor in an effective amount such that activity ofT-lymphocytes therein is substantially inhibited.
 35. The method ofclaim 34, wherein the tissue or organ transplant is selected from thegroup consisting of stem cells, bone marrow, heart, liver, kidney, lung,pancreas, small intestine, cornea, and skin.
 36. The method of claim 34,wherein the adenosine deaminase inhibitor is selected from the groupconsisting of pentostatin, fludarabine monophosphate, and cladribine.37. The method of claim 34, wherein the activity of T-lymphocytes in thetransplant is inhibited by at least 50%.
 38. The method of claim 34,wherein the activity of T-lymphocytes in the transplant is inhibited byat least 80%.
 39. The method of claim 34, wherein the activity ofT-lymphocytes in the transplant is inhibited by at least 90%.
 40. Themethod of claim 34, wherein treating the transplant includes storing thetransplant in a preservation solution containing pentostatin.
 41. Themethod of claim 40, wherein the preservation solution is Plegisol. 42.The method of claim 34, wherein treating the transplant includes washingthe transplant with a buffer containing pentostatin prior totransplantation.
 43. The method of claim 42, wherein the transplant isan isolated heart transplant.
 44. The method of claim 43, whereinwashing the heart includes flush perfusing the heart with the buffercontaining about 0.1-10 μM pentostatin.